ABSTRACT Modifying stem cells by insertion of anti-HIV gene or gene editing technologies can theoretically result in an HIV proof immune system in patients after stem cell transplantation. The success of the ?Berlin patient? receiving CCR5 negative cells for transplantation has demonstrated the feasibility of this approach. Thereafter, a myriad gene therapy trials targeting CCR5 to prevent HIV entry have been tried with inconclusive results. Recent, mathematical modeling data suggests that CCR5 disrupting strategies alone will fail to suppress HIV load unless combined with a suicide gene. Thus, there is an urgent need to develop new combination gene therapy vectors and methodologies that can be used to enrich vector modified stem cells before transplantation to ensure robust engraftment. Our proposal addresses these issues via a two-step vector system that incorporates multiple safety as well as selection features. Our vector 1 is a conditional vector that expresses the TK-SR39 gene only in the presence of HIV Tat protein. TK-SR39 is a TK mutant that is highly potent at killing cells in the presence of small amounts of Ganciclovir, a drug routinely used for Herpes virus treatment in HIV infected individuals. Furthermore, our vector incorporates an added unique feature that it also transiently expresses a Tat inducible modified CD4 (CD4mod) that is a CD4 molecule lacking the cytoplasmic domain as well as HIV Env binding region. This will provide a mechanism to purify modified cells using immunomagnetic beads while eliminating its use as a receptor for HIV entry. To induce transient expression of CD4mod we will use vector 2 that expresses HIV Tat and a CRISPR-CCR5 cassette to knockout CCR5. Thus, cells modified by this two-step vector system will have CCR5 knockout as well as conditional expression of TK-SR39 and CD4mod that will allow easy selection of cells. Thus, these cells encompass multiple layers of anti-HIV activity as 1) because of CCR5 knock out they will resist HIV infection 2) in case of CXCR4 virus emergence, the cytotoxic potential of TK-SR39 can be utilized via GCV administration 3) this will lead to rapid death of infected cells thereby minimizing virus spread and preventing establishment of latent reservoirs. The superiority of our approach lies in targeting the virus at multiple fronts and allowing a method to sort modified cells that is the key to long-term engraftment in vivo.